The present invention relates to devices and methods of use thereof for determining the presence and concentration of chemicals in a cell, tissue, organ or organism. The invention relates to, inter alia, semiderivative voltammetric measurements and chronoamperometric measurements of chemicals, e.g. neurotransmitters, precursors and metabolites, to identify, diagnose, and/or treat neuropathologies, neurotoxicities, tumors, and brain and spinal cord injuries.
Microvoltammetric indicator microelectrodes pass small but measurable currents while neurotransmitters and metabolites close to the microelectrode surface undergo oxidation and/or reduction (Adams R N et al., 1982, Handbook of Psychopharmacology, pp. 1–74). When an electrode is placed in contact with a solution a phase boundary is created that separates identical solutes into two different types. They are (a) molecules that are at a distance from the microelectrode and (b) those molecules that are close enough to participate in mutual interactions between the surface of the microelectrode and the sample solution interface (Kissinger P T et al., 1996, Laboratory Techniques in Electroanalytical Chemistry, pp. 11–50). Collectively, these interactions are called electrochemistry.
Detection of electrochemical signals from solutions and from anatomic brain sites is termed “faradaic” because the amount of the oxidative and/or reductive species detected at the surface of the microelectrode may be calculated by a derivation of Faraday's Law, the Cottrell Equation,
      i    t    =                    nFAC        0            ⁢              D        0                  1          /          2                                    3.14                  1          /          2                    ⁢              t                  1          /          2                    wherein i is current at time t, n is the number of electrons (eq/mol), F is Faraday's constant (96,486 C/eq), A is electrode area (cm2), C is concentration of oxygen (mol/cm3), and D is the diffusion coefficient of oxygen (cm2/s). The proportionality between charge and mass of an electrochemical reaction describes the relationship between the charge of each neurochemical in the process of oxidation and/or reduction and the concentration of each neurochemical. The Cottrell Equation relates to quiet solution experiments wherein the potential is instantaneously switched from an initial value Ei to a final potential, then held constant for a fixed time, then switched back to Ei. If material diffuses to a planar electrode surface in only one direction (linear diffusion) then the exact description of the current-time curve is the Cottrell Equation.
Current-time relationships with a circular electrode are defined in electrochemistry by the Cottrell Equation. For a long time, other electrode sizes and experiments using different electrolysis times were considered deviations from the Cottrell Equations that could be considered negligible. However, Wightman et al. observed that linear diffusion is not enough to describe the action that takes place at spherical microelectrodes (Dayton M A et al., 1980, Anal. Chem. 52:948–950). The quiet solution behavior of very small electrodes is different and is better described by a steady state equation in which the radius of the electrode is taken into account (Adams R N et al., 1982, Handbook of Psychopharmacology, pp. 1–74). This equation is suitable for calculating the edge effect or spherical steady-state contribution for even a 300-micron diameter electrode. Such a calculation reveals that the edge effect or spherical steady-state contribution adds approximately 30% current to the linear diffusion component for an electrolysis time of only one second (Dayton M A et al., 1980, Anal. Chem. 52:948–950).
Microvoltammetric circuits using several types of stearate-carbon paste microelectrodes have been developed and refined (Broderick P A, 1995, U.S. Pat. No. 5,433,710; Broderick P A, 1996, EP 90914306.7; Broderick P A, 1999, U.S. Pat. No. 5,938,903). Reliable separation and quantification of neurotransmitters including norepinephrine, serotonin, and dopamine as well as some of their precursors and metabolites is now possible (Broderick P A, 1989, Brain Res. 495:115–121; Broderick P A, 1988, Neurosci. Lett. 95:275–280; Broderick P A, 1990, Electroanalysis 2:241–245).
One electrode for in vivo electrochemical studies was developed in the laboratory of Ralph Adams (Kissinger P T et al., 1973, Brain Res 55:209). Using carbon paste electrodes with diameters reaching 1.6 mm and Ag/AgCl (3M NaCl) reference electrodes, neurotransmitters including dopamine and norepinephrine and their metabolites were detected (not separated), as a single peak in rat caudate nucleus with finite current electrochemistry and cyclic voltammetry.
Extensive refinements to microelectrodes and to in vivo electrochemistry have been made (Broderick P A, 1990, Electroanalysis 2:241–245). The recent development of a stearate-carbon paste probe along with an electrode conditioning process has resulted in reliable separation and detection of norepinephrine, dopamine, and serotonin (Broderick P A, 1996, EP 90914306.7; Broderick P A, 1999, U.S. Pat. No. 5,938,903). In addition, other types of microelectrodes with increased sensitivity and reliability continue to be developed (Broderick P A, 1996, EP 90914306.7; Broderick P A, 1999, U.S. Pat. No. 5,938,903). An electrochemically pre-treated carbon fiber electrode allows the differentiation of dopamine from DOPAC (Akiyama R A et al., 1985,Anal Chem. 57:1518), as do microelectrodes used in the instant invention.
Previous in vitro analysis techniques have yielded disappointing results. Prior ex vivo studies attempted to circumvent these problems with the microdialysis technique (During M J et al., 1993, Lancet 341:1607–1610; Lehmann A et al., 1991, Neurotransmitters and Epilepsy, pp. 167–180). Dialysis tubing placed on or within the brain is perfused with artificial CSF or Krebs-Ringer bicarbonate solution, and the perfusate is then analyzed with High Performance Liquid Chromatography (HPLC) with electrochemical detection; this provides information about the extracellular environment. However, this technique has been criticized because of the local gliosis caused by the dialysis probes and the perfusion process that can alter the biochemical parameters under study. In addition, the perfusate is analyzed outside the brain and therefore in contrast to microvoltammetry measurements are not truly in situ or in vivo.
Epilepsy is a neurological disorder characterized by transient electrical disturbances of the brain that may be studied by electrophysical techniques. Neurotransmitter data from experimental epilepsy models and in vitro analysis of surgically resected specimens from patients with partial epilepsy have thus far yielded conflicting results. These conflicting results may be due to significant variations between samples as well as choice of controls. Additionally, highly localized changes in epileptic cortex are not detectable using whole tissue homogenates. In general, increased activity in noradrenergic, dopaminergic, and serotonergic systems are believed to reduce cortical excitability and decrease seizure activity (Delgado-Escueta A V, 1984, Ann Neurol. 16(Suppl.): 145–148). However, human temporal lobe epilepsy is a complex disorder that may involve the dysfunction of distinct neuronal systems including the hippocampus and entorhinal cortex, the temporal neocortex, or combinations of these structures. Therefore, the contribution of different neurotransmitter systems to epileptogenesis in a given patient likely varies with lesion location and the etiology of epilepsy. Furthermore, recent studies demonstrating presynaptic inhibitory serotonin autoreceptors,-in hippocampus (Schlicker E et al., 1996, Naunyn Schmiedebergs Arch Pharmacol. 354:393–396) and a dual role for norepinephrine in epileptogenesis (Radisavljevic Z et al., 1994, International Journal of Developmental Neuroscience 12:353–361) suggest an even more complex situation.
Recent studies are now defining a syndrome of neocortical temporal lobe epilepsy that has distinct clinicopathologic and electrophysiologic features from mesial temporal lobe epilepsy (Pacia S V et al., 1997, Epilepsia 38:642–654; Pacia S V et al., 1996, Ann Neurol 40:724–730). While both mesial temporal lobe epilepsy and neocortical temporal lobe epilepsy are potentially treatable with surgical resection when seizures are refractory to antiepileptic medication, the type and extent of temporal lobe resection necessary to achieve a seizure free outcome may differ. Neocortical temporal lobe epilepsy patients may require resections tailored to include the epileptogenic zone. These resections may lie outside the boundaries of a standard temporal lobe resection performed for mesial temporal lobe epilepsy. Neurochemistry using microvoltammetry may provide a means for defining the epileptogenic zone in these patients.
Other techniques for detecting neurotransmitters in real time and in vivo fall short of the instant invention. These previous methods such as dialysis have limitations such as those described in During M J et al., 1993, Lancet 341:1607–1610; Ferrendelli J A et al., 1986, Adv. Neurol. 44:393–400; Goldstein D S et al., 1988, J Neurochem 50:225229; Janusz W et al., 1989, Neurosci Res 7:144153; Kawaguchi Y et al., 1998, J Neurosci 18:6963–6976.
In vivo detection of neurotransmitters and other chemicals is also important for diagnosing and treating movement disorders such as spinal cord injuries and brain injuries. Current techniques are limited, in part, in their relative inability to monitor neural chemistry in real time in a freely behaving animal or human which may limit their diagnostic and/or therapeutic efficacy. Movement may be generated by a central pattern generator (CPG), i.e. a neuronal network capable of generating a rhythmic pattern of motor activity either in the presence or absence of phasic sensory input from peripheral receptors.
Central pattern generators have been identified and analyzed in more than fifty rhythmic motor systems and CPG's can generate a variety of motor patterns. A universal characteristic of this wide variety of motor patterns is that they consist of rhythmic and alternating motions of the body or appendages. It is the rhythmicity of these behaviors that make these behaviors appear stereotypic. It is the repetitive quality of these behaviors that enables stereotypic behaviors to be controlled automatically. This automaticity or autoactivity means that there may be little or no need for intervention from higher brain centers when the environment remains stable.
The simplest CPG's contain neurons that are able to burst spontaneously. Such endogenous bursters can drive other motor neurons and some motor neurons are themselves, endogenous bursters. Importantly, bursters are common in CPG's that produce continuous rhythmic movement, such as locomotion. But, locomotion is an episodic, rhythmic behavior and thus, further regulation by neurochemicals becomes necessary. Endogenous bursts (cell firing) of neurons involved in locomotion must be regulated by neurotransmitters and neuromodulators, i.e., substances that can alter the cellular properties of neurons involved in CPG's. Brief depolarizations occur and lead to maintained depolarizations (plateau potentials) that can last for long periods of time. These maintained depolarizations far outlast the initial depolarization and it is these maintained depolarizations that are necessary for rhythmic movements. The generation of rhythmic motor activity by CPG's can be altered by amines and peptides (Grillner S et al., 1987, Trends Neurosci. 10:34–41; Rossignol S et al., 1994, Curr. Opin. Neurobiol. 4:894–902), thereby enabling a CPG to generate an even greater variety of repetitive motor patterns. Motor CPG's produce a complex temporal pattern of activation of different groups of motor functions and each pattern can be divided into a number of distinct phases even within a phase. CPG's are time-dependent (Pearson K et al., 2000, Principles of Neural Science, 4th edition, pp. 738–755).
Serotonin is an important neuromodulator for CPG's and can control the CPG underlying the escape swim response in the mollusc, Tritonia diomedea. The dorsal swim interneurons (DSI'S) are a bilaterally represented set of three 5-HTergic neurons that participate in the generation of the rhythmic swim motor program. Serotonin from these CPG neurons is said to function as both a fast neurotransmitter and as a slower neuromodulator. In its modulatory role, 5-HT enhances the release of neurotransmitter from another CPG neuron, C2 and also increases C2 excitability by decreasing spike frequency adaptation. Serotonin intrinsic to the CPG may neuromodulate behavioral sensitization and habituation. Serotonin intrinsic to the DSI enhances synaptic potentials evoked by another neuron in the same circuit (Katz P S, 1998, Ann. NY Acad. Sci. 860:181–188; Katz P S et al., 1994, Nature 367:729–731).
In another mollusc, the pteropod Clione limacina, the CPG for swimming is located in the pedal ganglia and formed by three groups of interneurons which are critical for rhythmic activity. The endogenous rhythmic activity of this CPG was enhanced by 5-HT (Arshavsky Y I et al., 1998, Ann. NY Acad. Sci. 860:51–69). In the pond snail, Lymnaea stagnalis, 5-HT is the main neurotransmitter in its stereotypic feeding circuit (Sadamoto H et al., 1998, Lymnaea Stagnalis. Neurosi. Res. 32:57–63). In the sea slug, Aplysia, the CPG for biting is modulated both intrinsically and extrinsically. Intrinsic modulation has been reported to be mediated by cerebral peptide-2 (cp-2) containing CB1–2 interneurons and is mimicked by application of CP-2, whereas extrinsic modulation is mediated by the 5-HT-ergic metacerebral cell (MCC) neurons and is mimicked by application of 5-HT (Morgan P T et al., 2000, J. Neurophysiol. 84:1186–1193).
In vertebrates, the 5-HT somatodendritic nuclei, the raphe, comprise the most expansive and complex anatomic and neurochemical system in CNS. Raphe nuclei almost exclusively reside along the midline in the rat and in the primate. Fewer reside along the midline, but several exhibit a paramedian organization (Azmitia E C, 1986, Adv. Neurol. 43:493–507). The rostral 5-HT raphe group and caudal linear nucleus sends 5-HT efferents to A9 basal nuclei motor systems and the caudal 5-HT group, whereas the interfascicular aspect of the 5-HTergic dorsal raphe projects efferents to A10 basal ganglia (nuclei) regions (Jacobs B L et al., 1992, Physiol. Rev. 72:165–229).
Electrophysiological studies have shown that the most prominent action of increased 5-HT cell firing, in 5-HT somatodendrites in treadmill locomotion for example, is to increase the flexor and extensor burst amplitude of 5-HT cell firing in dorsal raphe, (DR) somatodendrites for 5-HT, during locomotion (Barbeau H et al., 1991, Brain Res. 546:250–260). Further evidence for 5-HT controlling motor output is seen from studies in which 5-HT, directly injected into the motor nucleus of the trigeminal nerve, increased the amplitude of both the tonic electromyogram of the masseter muscle and the externally elicited jaw-closure (masseteric) reflex (McCall R B et al., 1979, Brain Res. 169:11–27; McCall R B et al., 1980, Eur. J. Pharmacol. 65:175–183; Ribeiro-Do-Valle L E et al., 1989, Soc. Neurosci. Abstr. 15:1283). In fact, Jacobs and Azmitia have proposed that 5-HT's primary function in CNS neuronal circuitry is to facilitate motor output (Jacobs B L et al., 1992, Physiol. Rev. 72:165–229).
Serotonin neurons within 5-HT somatodendrites depolarize with such extraordinary regularity that they exhibit automaticity, i.e., they can act by a CPG and produce plateau potentials. Thus, 5-HT neurons exhibit repetitive discharge characteristics. Increased 5-HT neuronal cell firing in somatodendritic raphe nuclei generally precedes the onset of movement or even increased muscle tone in arousal by several seconds and is maintained during sustained behavior (Jacobs B L, 1986, Neurochemical Analysis of the Conscious Brain: Voltammetry and Push-Pull Perfusion, Ann. NY Acad. Sci., pp. 70–79). Importantly, 5-HT cell firing in raphe nuclei is sometimes phase-locked to repetitive behavioral stereotypic responses. The regular firing of 5-HT somatodendrites in raphe nuclei is activated preferentially. This activation is associated with locomotion and chewing, stereotypic behaviors that are stimulated by CPG's (Jacobs B L et al., 1991, Pharmacol. Rev. 43:563–578). Serotonin intrinsic CPG's have been reported to be responsible for inducing rhythmic motor activity in the spinal cord of the turtle and the lamprey (Guertin P A et al., 1998, Neurosci. Lett. 245:5–8; Harris-Warrick R M et al., 1985, J. Exp. Biol. 116:27–46). The evidence in the lamprey suggests that 5-HT may have a role in the generation of a family of related undulatory movements, including, swimming, crawling, and burrowing, by a single CPG.
In addition to neurological disorders and injuries, the device and methods of use provided herein may be used for for brain cancer diagnosis and treatment. Current imaging technology is limited with respect to tumor visualization in neural tissue. For example, magnetic resonance imaging MRI is limited in its ability to detect tumor infiltration into white matter. This may hinder a physician's ability to render a diagnosis and/or prognosis. It further limits the ability to treat the patient by, for example, hindering a surgeon from defining tumor boundaries to remove the tumor. Alternatively, an inability to visualize cancerous cells or tissue in white matter may hinder a physicians ability to monitor the efficacy of a chemotherapy regimen.